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采用融合蛋白技术原核表达 CYP1 A1 (第 2 4 1- 381个氨基酸 )与谷胱甘肽 S-转移酶 (GST)的融合蛋白作为抗原 ,用于制备 CYP1 A1多克隆抗体 .根据正反重组质粒 p GEX/ 1 A1表达的融合蛋白大小不同的原理 ,直接表达筛选得到正向重组质粒p GEX/ 1 A1 .通过优化表达条件 ,提高了目的蛋白的表达水平 .包涵体蛋白经制备型聚丙烯酰胺凝胶电泳 (PAGE)分离 ,获含纯化融合蛋白 GST- 1 A1的 PAGE凝胶 .直接用含 GST- 1 A1的凝胶悬液免疫 BALB/ c小鼠 ,自腹水中获取 CYP1 A1多克隆抗体 (1 A1 p Ab) . 1 A1 p Ab用切胶纯化的融合蛋白GST- 2 B6交叉吸收 ,蛋白 A- Sepharose亲和层析柱来纯化 .用切胶纯化的融合蛋白 GST- 1 A1及 GST-2 B6的免疫印迹反应初步鉴定 1 A1 p Ab的特异性 .纯化的 1 A1 p Ab对融合蛋白 GST- 1 A1反应特异性较强 ,但仍对 GST- 2 B6有弱交叉反应 .在实际应用中可根据反应强度来加以区分
The fusion protein of CYP1 A1 (amino acid number 2-4 1-381) and glutathione S-transferase (GST) was used as antigen to prepare polyclonal antibody of CYP1 A1.According to the positive and negative recombinant plasmids pGEX / 1 A1 fusion protein expressed by different sizes, the direct expression of the plasmid was screened positive recombinant pGEX / 1 A1 by optimizing the expression conditions to improve the expression of the protein of interest. Inclusion body protein by the preparation of polyacrylamide Gel electrophoresis (PAGE) was used to isolate the PAGE gel containing the purified fusion protein GST-1 A1.The BALB / c mice were directly immunized with a GST-1 A1-containing gel suspension and the CYP1 A1 polyclonal antibody 1 A1 p Ab). 1 A1 p Ab was purified by gel electrophoresis of GST-2 B6 and protein A-Sepharose affinity chromatography.The purified GST-1 A1 and GST- 2 B6 Western blotting was used to identify the specificity of 1 A1 p Ab.The purified 1 A1 p Ab showed strong specificity for the fusion protein GST-1 A1 but still had a weak cross-reaction with GST-2 B6. According to the reaction intensity to be distinguished